1. Field of the Invention
The present invention relates to a separation method, a separation device and a separation apparatus for separating a body fluid such as lymph or blood into respective components and, more particularly, to a separation method, separation device and a separation apparatus for separating a body fluid into its respective components utilizing the difference in adherence of each body fluid component to hydrophilic groups of a lipid.
In a description to follow, the body fluid component is exemplified by lymphocytes and the present invention will be described with respect to separation of limphocytes into its fractions, i.e., T-cells and B-cells. However, the present invention is not limited thereto and can be widely adapted to separation of other body fluid components such as blood components or ascites components.
2. Description of the Prior Art
Lymphocytes play an important role in the immunological surveillance mechanism but are also associated with factors leading to diseases such as the Good syndrome. For this reason, in order to allow analysis of a biological defense mechanism or of interaction between cells or to allow clinical tests of an immunological deficiency syndrome, separation of lymphocytes into T-cells or B-cells or into its subsets having specific functions is an important task.
Cell populations called lymphocytes are known to be classified into at least two types having properties different in various respects. Typically, such cell populations are T-cells, i.e., thymus-derived lymphocytes, and B-cells, i.e., bone marrow-derived lymphocytes.
No conventional separation method of T- and B-cells allows complete separation of lymphocytes into T- and B-cells in one step. According to any conventional method of separating peripheral blood into T- and B-cells, in a first step, a leucocyte supernatant containing 70 to 90% of lymphocytes is separated from leucocytes by centrifugation using a high-density isotonic solution such as Ficoll-Paque, and the supernatant is then subjected to a second step. Six methods are mainly known as methods used in the second step:
(1) The rosette formation method using erythrocytes of sheep treated with neuraminidase
(2) The column method using nylon fiber
(3) The separation method using a water-insoluble, hydrophobic solid having a porous surface, or, a granular hydrophobic solid having acidic functional groups
(4) The separation method using a fluorescence cell sorter
(5) The continuous electrophoresis method using no carrier
(6) Affinity chromatography
However, the above methods have the following problems:
In the method (1), since the erythrocytes of sheep and lymphocytes are caused to exist in common, the lymphocytes are subject to stimulation.
The method (2) does not allow complete separation and cannot provide a satisfactory recovery rate of T-cells. In addition, no method is available for separating B-cells adhering to nylon fiber in a viable state.
Although the method (3) has a good separation effect and a good recovery rate of T-cells, it has only a low recovery rate of B-cells.
Since the method (4) uses an antiserum as a fluorescent label, the cells are subject to stimulation or damage. In addition, the method does not allow separation of a massive amount of lymphocytes such as about 10.sup.7 to 10.sup.8 lymphocytes.
The method (5) has an advantage of allowing separation of a massive amount of lymphocytes. However, mobility of cells differs depending upon their maturity, and the influence of the electric field on the cells has not yet been known.
The method (6) involves an unknown factor in respect of a change in functions of cells.
The methods (1) to (6) except for the method (5) commonly suffer from the problem of complex procedures or a long treatment time.
Although practical prior arts are disclosed as per Japanese Patent Disclosure Nos. 56-14088 and 58-74611, they both belong to one of the methods enumerated above and are therefore subject to the same problems.